Project description:Despite recent successes of precision and immunotherapies there is a persisting need for novel targeted or multi-targeted approaches in complex diseases. Through a systems pharmacology approach including phenotypic screening, chemical and phosphoproteomics and RNA-Seq, we elucidated the targets and mechanisms underlying the differential anticancer activity of two structurally related multi-kinase inhibitors, foretinib and cabozantinib, in lung cancer cells. Biochemical and cellular target validation using probe molecules and RNA interference revealed a polypharmacology mechanism involving MEK1/2, FER and AURKB, which were each more potently inhibited by foretinib than cabozantinib. Based on this, we developed a synergistic combination of foretinib with barasertib, a more potent AURKB inhibitor, for MYC-amplified small cell lung cancer. This systems pharmacology approach showed that small structural changes of drugs can cumulatively, through multiple targets, result in pronounced anticancer activity differences and that detailed mechanistic understanding of polypharmacology can enable repurposing opportunities for cancers with unmet medical need.
Project description:Isolated murine kidney glomeruli were lysed in buffer containing 8M urea, and proteins were reduced and alkylated. Proteins were digested with trypsin. After reverse-phase chomatrography, peptides were fractionated using strong cation exchange chromatography. Phosphopeptides were enriched using IMAC (FeNTA) columns. Peptides were analyzed by LC-MS/MS on a Q Exactive mass spectrometer or an LTQ Orbitrap XL mass spectrometer. Metadata for Orbitrap samples (MaxQuant Output files include „Orbi“) 1. General features – 1.1 Global descriptors a. Responsible person or role: Markus Rinschen. markus.rinschen@uk-koeln.de; tobias.lamkemeyer@uni-koeln.de b. Instrument manufacturer, model: LTQ-Orbitrap XL, Thermo Scientific c. customization 2. Ion sources – ESI fed by nLC 2 (Proxeon) 3. Post source component 3.1. Analyser: MS1 survey scan in an Orbitrap and MS2 analysed in Linear Trap. 3.2. Activation/Dissociation: CID 4. Spectrum and peak list generation and annotation 4.1. Data acquisition: MaxQuant v. 1.3.05 Top one method with a cycle of one full MS1 scan in the Orbitrap, followed by the fragmentation with dynamic exclusion window of 60 seconds and followed by the acquisition of 5 product ion scans generated in the LTQ analyser, and detected in the LTQ. Unselected fragmentation. For further details, see .raw files. URL of file: Filename 4.2. Data analysis: MaxQuant v 1.3.05 Parameters used in the generation of peak lists or processed spectra: see annotation in “parameters” file. The mouse uniprot reference database “complete proteome” obtained on January 2nd, 2013 was used. Metadata for QExactive samples (Max Quant output files include „QE“) 1. General features – 1.1 Global descriptors a. Responsible person or role: Markus Rinschen. markus.rinschen@uk-koeln.de; Marcus Krüger marcus.krueger@mpi-bn.mpg.de b. Instrument manufacturer, model: Q Exactive Thermo Scientific c. customization 2. Ion sources – ESI fed by nLC 1000 (Proxeon) 3. Post source component 3.1. Analyser: MS1 survey scan in an Orbitrap and MS2 analysed in Orbitrap/HCD cell 3.2. Activation/Dissociation: HCD 4. Spectrum and peak list generation and annotation 4.1. Data acquisition: MaxQuant v. 1.3.05 Top one method with a cycle of one full MS1 scan in the Orbitrap, followed by the fragmentation with dynamic exclusion window of 20 seconds in the HCD cell and followed by the acquisition of 10 product ion scans generated in the LTQ analyser, and detected in the LTQ. Unselected fragmentation. For further details, see .raw files. URL of file: Filename 4.2. Data analysis: MaxQuant v 1.3.05 Parameters used in the generation of peak lists or processed spectra: see annotation in “parameters” file. The mouse uniprot reference database “complete proteome” obtained on January 2nd, 2013 was used.
Project description:Covalent inhibitors of KRASG12C (KRASi) hold considerable progress for tumors driven by this oncogene yet early studies suggest rapid mechanisms of adaptive resistance that appear cell type dependent. To address these challenges, we performed mass spsectrometry baesd phosphoproteomics analysis in KRASG12C cell lines after short term treatment with ARS-1620 and compared phosphoproteomes of KRASG12C cells to understand signaling diversity. Our analysis suggests individual KRASG12C cells responds uniquely to perturbation of KRASG12C. Cell line models can be categorized in epithelial or mesenchymal subtypes and a similar pattern was observed in KRASG12C human lung cancer tumor tissues. ERBB2/3 signaling compensate for repressed ERK signaling following ARS-1620 treatment in epithelial cell type, and this subtype was also more responsive to inhibition of SHP2, IGFR, and SOS1. Conversely, FGFR signaling drives resistance to KRASi in mesenchymal cells in part via mTOR signaling. These studies suggest transcriptional subtypes of KRASG12C dictate responsiveness to specific combinations.
Project description:This project includes two separate experiments where the ubiquitylated proteome of the budding yeast Saccharomyces cerevisiae was enriched from whole cell lysate by affinity purification using a novel OtUBD affinity resin. In the first experiment, two distinct experimental conditions (native, denaturing) were used to compare the proteins enriched by OtUBD affinity resin and the negative control resin. In the second experiment, the ubiquitylation profile of proteins were compared among wildtype yeast cells and yeasts lacking certain E3 ligase enzymes.
Project description:The motivation for the present study was to generate a novel data set, GeneNote (Gene Normal Tissue Expression), in order to portray the complete picture of gene expression profiles in healthy human tissues. We used the complete Affymetrix GeneChip HG-U95 set, which includes 62,839 probe-sets, representing a nearly complete set of human genes. The intensity results of two replicates were processed and analyzed to yield the complete transcriptome for the following human tissues: brain, spinal cord, heart, skeletal muscle, bone marrow, thymus, lung, liver, spleen, pancreas, prostate and kidney.